The present invention relates to apparatus and methods for the development of an embryo outside the body, and more specifically, to apparatus and methods of providing the optimal environment for embryo development until the embryo is ready for implantation into the body.
About every 28 days or so, the post-pubescent female human goes through the reproductive cycle. The cycle is divided into two phases: the follicular phase (generally the first 14 days, or half of the cycle); and the luteal phase (generally the last 14 days, or half of the cycle).
During the follicular phase, the anterior pituitary gland secretes follicle stimulating hormone (FSH), which is a small glycoprotein. The ovaries have a specific receptor cite for the FSH. FSH assists in the development of one or two small cysts (i.e., egg follicles) in the ovaries, each of which contains an ovum. Cells surrounding the developing ovum, in turn, produce estrogen. Estrogen has several effects on the body during the follicular phase. First, it stimulates development of the endometrium: the velvet-like interior lining of the uterus which allows the uterus to receive and support an embryo. Secondly, estrogen regulates the release of FSH from the anterior pituitary gland. At low levels, estrogen modulates the release of FSH. However, at higher levels, estrogen provides a positive feedback on the pituitary gland, inhibiting the release of FSH and stimulating the release of luteinizing hormone (xe2x80x9cLHxe2x80x9d).
LH is released from the anterior pituitary gland on about day 13 of the reproductive cycle. LH assists in causing ovulation: the release of an ovum (i.e., egg) from its follicle. Distal fingers or frimbrae of a fallopian tube embrace or pick up the ovum and envelope it in the distal portion of the fallopian tube, also known as the ampullae.
The ampullae is an about 2 to about 2.5 cm tubal segment of the fallopian tube having a diameter of about 1 to about 2 cm. Fertilization (union of the capacitated sperm and ovum) occurs in this portion of the fallopian tube. After fertilization, the. fertilized ovum (or embryo) slowly migrates along the fallopian tube towards the uterus. The embryo spends its first 2 to 3 days in the ampullae where the embryo (commonly referred to as a zygote at this stage of development) rapidly divides into a ball of cells.
The interluminal environment of the fallopian tube consists of a serum transudate, which is produced from the epithelial cell lining of the fallopian tube""s lumen. A rich vascular supply exists along the entire length of the fallopian tube, with collateral circulation from both the uterine and ovarian arteries and veins. The serum transudate establishes an equilibrium with the epithelial cell arterioles and capillaries to supply nutrients, glucose, amino acids, and oxygen to the developing embryo in the fallopian tube. Moreover, metabolic waste, including carbon dioxide, is evacuated from serum transudate by diffusion into the capillaries. The constant supply of nutrients, glucose, amino acids, and oxygen to the developing embryo and the rapid elimination of metabolic waste, including carbon dioxide, provides an optimum environment for embryo development in the fallopian tube.
There are generally two methods of transportation of the embryo through the fallopian tube. First, the fallopian tube contracts as a muscle to move the embryo along its length towards the uterus. Second, fallopian tube epithelial cell cilia assist in moving the ovum or embryo from the ovary to the uterus. In fact, the cilia (hair like projections) create a current in the serum transudate. Both the muscle contraction and the cilia movement create a xe2x80x9cto-and-froxe2x80x9d or xe2x80x9cback-and-forthxe2x80x9d movement within the fallopian tube. This movement of the serum transudate (i.e., fluid in the fallopian tube) and embryo creates an intraluminal circulation system which assists in distributing nutrients and oxygen to the embryo, and removing metabolic waste (including urea and carbon dioxide) away from the embryo.
After ovulation, the reproductive cycle enters the luteal phase, wherein the ovaries secrete progesterone at the cite of ovulation. The cite of ovulation on the ovary is yellow and is commonly referred to as the corpus luteum. Progesterone stops the estrogen-mediated growth of the endometrium, and maintains the endometrium so as to prepare it for the reception and support of the developing embryo.
After fertilization, the embryo begins its migration toward the uterus. At first, the rate of travel is slow and the distance of travel is short. For example, about one day after fertilization, the embryo has traveled about 1 cm through the fallopian tube toward the uterus. The rate of travel and distance traveled increases as time after fertilization elapses, and as the number of cells increases. For example, the embryo may travel another cm between day 1 and 2 post fertilization, and between day 2 and 3 post fertilization. However, the rate and distance of travel increases whereby the embryo may travel 3 cm between day 4 and 5 post fertilization (See, e.g., FIGS. 4-5).
As the embryo migrates toward the uterus, it leaves the ampullae and enters the isthmus, the longest section of the fallopian tube at about 4 to 7 cm. The isthmus has both circular and longitudinal muscles which assist in the migration of the embryo toward the uterus. The embryo spends about 40-60 hours migrating through the distal portion of the isthmus, and about 15-20 hours migrating through the proximal portion of the isthmus. Thereafter, the embryo passes through the cornu and interstitial regions of the fallopian tube, taking about 3-4 hours to do so. As the embryo""s cell number increases from about 2 at day one post fertilization to about 32 at five days post fertilization (see, e.g., FIG. 6), its transit rate through the fallopian tube also increases. The increased transit rate is believed to assist in providing or making available additional nourishment to the embryo, and also to increase the rate of diffusion of waste products away from the embryo.
At about five days post fertilization (i.e., about 19 days into the reproductive cycle), the embryo enters the uterus. At this stage, the embryo is generally referred to as a blastocyst. The blastocyst may penetrate the endometrium whereby it implants and attaches to the uterine wall. At the point of implantation, the blastocyst divides into two distinct cell lines: the placental line, which will eventually develop into the placenta which assists in nourishing the fetus; and the fetal line. The placental line produces human chorionic gonadotrophin (HCG), which acts to continue the ovarian corpus luteum""s production of progesterone for about 11 to 12 weeks (until the placenta has sufficient progesterone to continue the pregnancy).
Many women cannot become pregnant for a variety of reasons. For example, occlusion or dysfunction of the fallopian tubes may lead to fertility problems. One of the traditional solutions to infertility has been adoption. However, the number of healthy infants available for adoption relative to the number of people seeking to adopt has substantially decreased in recent years. As such, adoption often is not readily available to every person who wishes to adopt a child.
Alternatively, there have been attempts in the past to restore normal tubal function to occluded or dysfunctioning fallopian tubes. Surgeons have tried to repair or reconstruct damaged fallopian tubes using surgery. In addition, physicians have also tried transplanting healthy fallopian tubes from a donor. There are several drawbacks with this course of treatment. First, major surgery can be required under a general anesthetic. Second, with regard to transplant, there is the possibility of open rejection by the recipient.
Another solution to a dysfunctional fallopian tube has been to implant an artificial fallopian tube in the woman""s body. An example of this is disclosed in U.S. Pat. No. 4,574,000 (Hunter). The apparatus includes a ovisac which encapsulates one of the ovaries in order to collect any ova discharge. Fluid supply tubes wash the ova toward a tubular member that is secured in communication with the uterine cavity. A reservoir of fluid and a programmable micropump are also provided, and are adapted to be implanted into the patient. The artificial fallopian tube, however, has several drawbacks. First, a patient""s natural fallopian tube must be excised and the artificial fallopian tube inserted in its place. Such a procedure requires major surgery with a general anesthetic to implant. In addition, fluid used to wash the ova toward the tubular member and the uterine cavity must be injected into the reservoir through the skin using a syringe and needle, as needed.
Another alternative for the dysfunctioning fallopian tube is to totally bypass damaged fallopian tubes and use an in vitro fertilization (xe2x80x9cIVFxe2x80x9d) technology (which is often referred to as xe2x80x9ctest tube babiesxe2x80x9d). This delicate procedure involves surgically removing a mature egg (an ovum) immediately prior to ovulation and placing it in a nutrient medium containing sperm. The sperm then fertilizes the ovum. Every 24 hours, the media is changed, generally by skilled embryologists, technicians, or physicians who physically move the embryo to another petri dish with fresh IVF media.
When in vitro fertilization was developed about 20 years ago, the embryo was transferred back to the body about 24 hours after fertilization. Since that time, the timing of the embryo""s transfer back into the body has increased from 1 day after fertilization to about 3 days after fertilization. There are, however, several drawbacks with this procedure and the equipment used.
At one to three days after fertilization, the embryo has not sufficiently matured so that it is ready for attachment and implantation to the endothelium of the uterus. One solution to this has been to insert the embryo into the portion of the fallopian tube where it would typically be located at three days after fertilization. This procedure is commonly referred to as Zygote, Intra Fallopian Transfer or ZIFT. Then, the fallopian tube should transport the embryo to the uterus and allow it to develop inside the body (in vivo) and the fallopian tube over the next several days. However, this procedure has certain drawbacks. For example, it has increased the risk of a tubal pregnancy, which is where the embryo implants in the fallopian tube. Such a pregnancy cannot continue and has to be terminated. If this condition or type of pregnancy is not detected, the growing embryo can damage and even rupture the fallopian tubes.
At day three of embryo development in an IVF procedure, the embryo often is too young to properly evaluate. More specifically, the embryo has not developed sufficiently or for a period of time whereby its progress can be tracked and charted to predict a successful pregnancy. As such, the embryo may not be sufficiently developed or had its progress charted so as to predict whether or not the embryo will result in a pregnancy, or whether the embryo has a lethal defect due to genetics or a toxin.
In order to improve the likelihood of a successful pregnancy, multiple embryos (as many as 4 or 5) may be inserted into the uterus at one time in the hope that one embryo will attach to the endometrium. However, in many cases, more than one embryo, and sometimes all of the embryos, attach to the endometrium, which creates a multiple embryo pregnancy. Multiple birth pregnancies have a higher risk for the mother and the embryos as compared to a single embryo pregnancy. Concerns and problems can include increased chances of premature delivery, lower birth weight babies, toxemia in the mother, and twin to twin transfusion. Even when multiple embryos are inserted, the success rate as measured by the percentage of implantations, and thus pregnancies, has been relatively low, at about 6-7% per ovum transferred.
Previously, the ovum was fertilized in a petrie dish and allowed to develop in another petrie dish. As the needs of the embryo changed, it was physically removed from a first petrie dish and placed in another petrie dish with new fluid. In addition, in order to inspect the embryo to monitor and record its development, the embryo would have to be manipulated with a pipette. As the embryo remained in the petrie dish, the media was generally stagnant and did not move or even swirl around the embryo.
This procedure has several drawbacks. First, it can be costly since it involves the use of special laboratories and skilled technicians to maintain the developing embryo. Second, fluid within the petrie dish remains stagnant and a circulation system for distribution of nutrients and removal of waste products is not present. Moreover, the low success rate, as measured in successful pregnancies, may necessitate multiple attempts at embryo implantation. These additional attempts can further increase the cost.
Recently, there has been a teaching that an embryo can be maintained in vitro for four to five days after fertilization (i.e., until the embryo reaches the blastocyst stage) and then inserted into the uterus for implantation. Dr. D. K. Gardner reported the use of a serum-free media, and the use of a different liquid media for the period exceeding 2-3 days after fertilization. The teachings and techniques of Dr. Gardner, however, do not address the concerns of circulating media around the embryo while in vitro, and/or eliminating manipulation of the embryo while in vitro.
As can be seen, currently available equipment and techniques have a number of shortcomings that can greatly reduce the ability of the embryo to develop in vitro and thus, the success rate for in vitro pregnancies. The current structures and assemblies provide a petrie dish and other equipment that require physical manipulation of the embryo, and moving it from one petrie dish to another to change the media solution. A need currently exists in the fertility medicine field for equipment and techniques to enhance development of an embryo in vitro before being implanted in the body, which increases the chances or opportunity for pregnancy to occur when in vitro fertilization is used. As such, the chances for pregnancy increase when in vitro fertilization is used.
It is an object of the present invention to provide apparatus and techniques which address and overcome the above-mentioned problems and shortcomings in the fertility medicine field.
Another object of the present invention is to provide apparatus and techniques which mimic the physiology of the internal fallopian tube.
Still another object of the present invention is to provide apparatus and methods which allow for the replacement of the IVF media without the need to manipulate the embryo.
Yet another object of the present invention is to provide apparatus and methods which change the embryo growth conditions as the metabolic needs of a developing embryo change.
A further object of the present invention is to provide apparatus and methods which permit visualization of the developing embryo, without physically manipulating the embryo.
Yet another object of the present invention is to provide apparatus and methods which permit photo documentation of the developing embryo.
Another object of the present invention is to provide apparatus and methods which allow for the sequential monitoring of the embryo.
Still another object of the present invention is to provide apparatus and methods which allow development of multiple embryos in vitro, and selection of one or more of the embryos for implantation into the female.
A further object of the present invention is to provide apparatus and methods that can be used with genetic engineering or animal husbandry.
It is yet another object of the present invention is to provide apparatus and methods that permit the growth and development of the embryo to be monitored and charted.
Additional objects, advantages and other features of the invention will be set forth and will become apparent to those skilled in the art upon examination of the following, or may be learned with practice of the invention. It should also be understood that the objects specifically identified above may or may not be provided by each and every embodiment of the present invention. Thus, these objects of the present invention are not to be construed as limiting in any way the scope of the claims appended hereto.
One embodiment of the present invention comprises a device for the in vitro development of an embryo in a fluid that includes a chamber having a tank receiving the fluid. The chamber has an container for housing the embryo and a circulator therein for the circulation of fluid, or exterior for movement of the chamber and fluid therein. At least one fluid reservoir is in fluid communication with the chamber and a collector reservoir can also be in fluid communication with the chamber. In a preferred embodiment of the present invention, the chamber may include at least one inlet port provided in the lower portion of the chamber, and/or an outlet port provided in the upper portion of the chamber.
The container can include at least one window whereby a visualization assembly can monitoring the inside of the container through the window or within the chamber. In a preferred embodiment, two windows may be oppositely disposed in the sides of the container. To permit or enhance the circulation of fluid around the embryo, the container may be made from a microporous material, preferably having a pore size of less than about 75 microns.
The present invention may also include a visualization assembly to monitor, visualize, and/or record the development and progress of the embryo, and/or the ovum and sperm before fertilization. The visualization assembly can include a viewing device, such as a magnification device to enhance the view of the contents, and/or a photodocumentation device, such as a camera. Additionally, the visualization assembly may also include a display device that can preferably be located away or remote from the chamber, or a printer.
The present invention can also include a sensor system in the chamber for monitoring the condition of the fluid. The sensor system can include different types of sensor, such as a pH sensor, an oxygen sensor, a thermometer, or a carbon dioxide sensor.
The present invention may also include a feedback control system that can have a microprocessor; and a sensor system in electrical communication with the microprocessor, a pump in electrical communication with the microprocessor, a valve in electrical communication with the microprocessor, a temperature regulator in electrical communication with the microprocessor, or a circulator in electrical communication with the microprocessor.
The present invention also includes a method for the in vitro development of an embryo in a fluid, preferably until it reaches the blastocyst stage. An embryo or sperm and an ovum can be placed in container that is placed in a chamber for holding the fluid. Fluid within the chamber is circulated. Fluid is then selectively exchanged in the chamber. Exchanging the media can include removing fluid from the chamber to the collection reservoir, and inserting fluid from the reservoir into the chamber. This can occur simultaneously. Conditions of the fluid, such as pH levels, temperature, oxygen levels, or carbon dioxide levels can be monitored and displayed by a sensor system in the fluid.
Fluid in the chamber can be exchanged at predetermined time interval, of if the conditions in the fluid warrant a change. Examples of such condition changes may include temperature, oxygen, or carbon dioxide levels above or below prescribed levels.
Development and progress of the ovum, sperm and embryo can be monitored, viewed and recorded using a visualization assembly.
After the embryo has sufficiently developed, preferably to the blastocyst stage, it is transferred from the device to uterus of a female.
The present invention also provides a method for the in vitro development of an embryo, comprising:
(a) providing a tank having an embryo and a first fluid therein;
(b) monitoring the growth of the embryo; and
(c) adjusting conditions within the tank in response to the results of the monitoring step.
The step of monitoring the growth of the embryo may comprise, for example, monitoring the nuclear mass (i.e., cell count) of the embryo, such as by measuring the optical density of the embryo. The optical density of the embryo may be measured by directing light at the embryo, and measuring the amount of light transmitted through the embryo.
The step of adjusting conditions within the tank may comprise at least one of: adjusting the fluid pressure within the tank, flowing fluid into the tank and adjusting the temperature within the tank. By way of example, the fluid pressure within the tank may be increased as the nuclear mass of the embryo increases, thereby better simulating the conditions within a natural fallopian tube during transit of the embryo towards the uterus. The tank may include a fluid outlet, and the step of adjusting conditions within the tank may comprise adding new fluid to the tank while allowing the first fluid already in the tank to be removed from the tank through the fluid outlet. The new fluid may be the same as or different from the first fluid already in the tank. The step of adjusting conditions within the tank may also comprise replacing the first fluid with a second fluid after the nuclear mass of the embryo has reached a predetermined level, wherein the replacing step is accomplished without manipulating the embryo.
A plurality of tanks, each having an embryo and a first fluid therein, may also be provided. The step of monitoring the growth of the embryos may comprise monitoring the rate of growth of each embryo over a period of time, such that one or more of the embryos may be selected for insertion into a recipient based upon the rate of growth of those embryos (e.g., those embryos which grew at the optimal rate are chosen for implantation).
The present invention also provides a method for the in vitro development of an embryo, comprising:
(a) providing a tank having an embryo therein, the tank in fluid communication with at least first and second sources of fluid;
(b) flowing fluid from at least the first fluid source into the tank; and
(c) thereafter, flowing fluid from at least the second fluid source into the tank.
The tank may have a fluid inlet and a fluid outlet, such that fluid is urged out of the tank through the fluid outlet as fluid is flowed into the tank through the fluid inlet. Fluid may be continuously flowed into the tank (e.g., gravity fed, gas pressure fed or pumped), or may be periodically flowed into the tank (e.g., pulsed flow, or even flowed into the tank at predetermined intervals dependant upon a predetermined schedule, embryo growth, a sensed condition within the tank, and/or a sensed condition of fluid urged out of the tank). In addition, the step of flowing fluid from at least the second fluid source into the tank may similarly commence in response to at least one of: a predetermined schedule, the nuclear mass of the embryo, a sensed condition within the tank, and a sensed condition of fluid urged out of the tank.
The present invention further provides a method of fertilizing an egg and for the in vitro development of the resulting embryo, comprising:
(a) providing a tank having a fluid outlet and a fluid inlet;
(b) inserting an unfertilized egg, a fluid, and sperm into the tank;
(c) after the egg has been fertilized or after a predetermined period of time sufficient to allow the egg to be fertilized, flowing additional fluid into the tank through the fluid inlet, such that sperm and fluid already in the tank is urged out of the tank through the fluid outlet; and
(d) allowing the fertilized egg to develop in the tank.
A cartridge for use in the in vitro development of one or more embryos is also provided by the present invention, and comprises:
(a) a cartridge body; and
(b) at least one tank for housing an embryo therein, the tank having a fluid inlet and a fluid outlet;
wherein the cartridge is configured such that it may be placed in fluid communication with at least one fluid source such that fluid from the fluid source may be delivered to the tank through the fluid inlet, and the cartridge is further configured such that fluid may be removed from the tank through the fluid outlet. The cartridge may even comprise a plurality of the tanks, each of the tanks having a fluid inlet and a fluid outlet. The cartridge body may include one or more cartridge fluid inlets, each of which is in fluid communication with the fluid inlet on the tank.
The tank may include an upper portion and a lower portion, wherein the diameter of the upper portion is greater than the diameter of the lower portion. The fluid outlet may be located on the lower portion of the tank, and the fluid inlet located on the upper portion of the tank. The lower portion of the tank may also include a porous wall adjacent the fluid outlet, the porous wall configured to allow fluid (and even sperm and cellular debris) to pass therethrough, while preventing an embryo from passing therethrough. The tank may also have a port configured for inserting an embryo therethrough, as well as a valve in fluid communication with the fluid outlet. The cartridge body may include a cartridge fluid outlet in fluid communication with the fluid outlet on the tank.
The present invention also provides a system for the in vitro development of one or more embryos, comprising:
(a) a main housing for incubating at least one embryo therein, the housing configured for receiving a cartridge therein;
(b) a cartridge positioned in the main housing, the cartridge having at least one tank configured for housing an embryo therein; and
(c) one or more fluid sources for containing a fluid therein, at least one of the fluid sources in fluid communication with the at least one tank.
It should be pointed out that the phrase xe2x80x9cfluid communicationxe2x80x9d includes the situation wherein a valve or other flow control member is interposed between the two items which are in fluid communication in order to control (and even prevent) the flow of fluid therebetween. The cartridge may have a plurality of tanks, each of which is configured for housing an embryo therein, and wherein the at least one fluid source is in fluid communication with the tanks.
The system mat further comprise at least one pump for urging fluid from the at least one fluid source into the at least one tank, as well as at least one valve for regulating the flow of fluid from the at least one fluid source into the at least one tank. A processor (e.g., a CPU of the type used in general purpose or specialized computing devices) for controlling the flow of fluid from the at least one fluid source into the at least one tank may also be provided. The system may also include a visualization system for acquiring an image of an embryo positioned within the tank, as well as a display screen configured for displaying an image acquired by the visualization system. The visualization system may comprise, for example, a camera configured for acquiring an image of an embryo positioned within the tank. In one embodiment, images of an embryo positioned within the tank may be acquired at predetermined intervals, and even automatically as controlled by the processor. The visualization system may further comprise one or more fiber optic bundles positioned within the cartridge, the fiber optic bundle configured for transmitting an image of an embryo positioned within the tank to the camera.
A control system for regulating fluid conditions within the tank may also be included, and may comprise a processor, at least one sensor in electrical communication with the sensor, and at least one processor-controlled device chosen from the group consisting of: a heater, a pump configured for urging fluid from the at least one fluid source into the at least one tank, and at least one valve for regulating the flow of fluid from the at least one fluid source into the at least one tank. The control system may further include at least one alarm responsive to an electrical signal from the processor.
The system may also include a waste fluid reservoir in fluid communication with the at least one tank, as well as a plurality of the cartridges.